The production of fuel substitutes from renewable resources has gained significant attention because of the rising energy price, environmental concerns and the need to reduce dependence on fossil-derived transportation fuels. Currently, ethanol is the major form of biofuel, with 84 billion liters of bioethanol produced in the world in 2011. While both the production capacity and the demand for bioethanol are increasing rapidly, ethanol properties are incompatible with existing fuel infrastructure. Indeed, ethanol's tendency to absorb water poses distribution problems in currently used pipelines and its low energy density (30% lower than gasoline) requires vehicle retrofitting in the fuel system when using high percentage blends with gasoline (Yan & Liao, 2009).
These problems with ethanol hinder large-scale replacement of gasoline. As an alternative, production of higher chain alcohol biofuels (n-propanol, n-butanol, isobutanol, methyl butanol), fatty acid esters and isoprenoids from renewable sources are of increasing interest because of their high energy densities and their low hygroscopicity, which reduce problems in storage and distribution and allow usage in current engines. However, these biofuel compounds with high fuel-quality are not commonly produced biologically (except, for instance, by some Clostridium species) and/or in large enough quantities for fuel applications.
1-Propanol (CH3CH2CH2OH; n-propanol, propan-1-ol, propylic alcohol, n-Propyl alcohol, Propyl alcohol, Propylol, Ethylcarbinol, 1-Hydroxypropane, Propionic alcohol, Propionyl alcohol, Propionylol) is an important industrial chemical that has been used as a major component of resins and as a carrier and extraction solvent in the pharmaceutical, paint, cosmetic (lotion, soap, and nail polish) and cellulose ester industries. It also has high biofuel potential in terms of combustion efficiency, storage convenience and transportation with an energy density and a flashpoint higher than methanol and ethanol.
Production and uses of 1-propanol are associated with its transformation into compounds such as propionic acid, iso-propanol, propionaldehyde and trihydroxymethyl ethane, all of which are important chemical commodities. Hundreds of thousands of tons of 1-propanol are produced by a two-step process requiring the catalytic hydroformylation of ethylene to produce propanal and then catalytic hydrogenation of the propanal. Alternatively, 1-propanol can also be produced as a by-product of fermentation of potatoes, but unlike ethanol and butanol, very few “green” biofermentation processes exist for the production of this very important commodity.
To circumvent these production issues, metabolic engineers have used genomic information and molecular biology techniques to construct user-friendly, heterologous (non-native) host organisms such as Escherichia coli or Saccharomyces cerevisiae to serve as a production platform for the production of fuel-grade compounds beyond the scope of what native organisms can produce. Microbial production of 1-propanol has been demonstrated by Clostridium sp. and yeast (Eden et al., 2001; Janssen, 2004), with final titers achieved less than 70 mg/L. Recently, a metabolically engineered Escherichia coli strain harboring 2-keto acid decarboxylase and alcohol/aldehyde dehydrogenase capable of producing 1 g/L of 1-propanol via 2-ketobutyrate was developed (Shen & Liao, 2013).
The introduction of a modified Methanococcus jannaschii citramalate synthase (encoded by cimA) that can directly convert pyruvate to 2-ketobutyrate, led to the production of up to 3.5 g/L of 1-propanol (Atsumi & Liao, 2008) (Howell et al., 1999). Thermobifida fusca, a cellulolytic microorganism, harboring the Clostridium acetobutylicum ATCC 824 alcohol/aldehyde dehydrogenase also produced 0.48 g/L of 1-propanol from untreated lignocellulosic biomass as a carbon source via 2-ketobutyrate as a metabolic intermediate (Deng & Fong, 2011).
Recently, a wild-type E. coli harboring 1,2-propanediol dehydratase from Klebsiella oxytoca was shown to produce 0.25 g/L of 1-propanol following additional engineering of the 1,2-propanediol pathway (Jain & Yan, 2011). Finally, production of 1-propanol through an amino acid biosynthetic pathway using glucose or glycerol as a carbon source was achieved in an E. coli strain engineered to establish a novel pathway leading to the formation of 1-propanol under aerobic condition and carrying plasmid-based atoDA, adhEmut, thrABC, ackA and cimA genes was able to produce more than 10 g/L of 1-propanol from glucose or glycerol in aerobic fed-batch fermentation (Shen & Liao, 2013, Choi et al., 2012, Shen & Liao, 2008) (Srirangan et al., 2013).
There is a need in the art for efficient means to produce 1-propanol. This invention provides methods, bacterial cultures, and systems to meet this and other needs.